Methods and compositions for treating neurodegenerative diseases

ABSTRACT

The present invention provides methods and compositions for treating neurodegenerative diseases by cyclohexenone compounds.

CROSS REFERENCE

This application claims the benefit of U.S. provisional application Ser.No. 61/729,295, filed Nov. 21, 2012, which is incorporated by referencein its entirety.

BACKGROUND OF THE INVENTION

Neurodegeneration is the umbrella term for the progressive loss ofstructure or function of neurons, including death of neurons. Manyneurodegenerative diseases including Parkinson's disease, Alzheimer'sdisease, and Huntington's disease occur as a result of neurodegenerativeprocesses. Neurodegeneration can be found in many different levels ofneuronal circuitry ranging from molecular to systemic.

Alzheimer's disease (AD) is characterised by loss of neurons andsynapses in the cerebral cortex and certain subcortical regions. ADindividuals show 70% loss of locus coeruleus cells that providenorepinephrine. Norepinephrine, in addition to its neurotransmitterrole, locally diffuses from “varicosities” as an endogenousantiinflammatory agent in the microenvironment around the neurons, glialcells, and blood vessels in the neocortex and hippocampus. It has beenshown that norepinephrine stimulates mouse microglia to suppressβ-amyloid (Aβ)-induced production of cytokines and their phagocytosis ofAβ. It is estimated that about 18 million people worldwide have AD.

SUMMARY OF THE INVENTION

In one aspect provided herein are methods for treating or reducing therisk of neurodegenerative disease comprising administering to a subjecta therapeutically effective amount of a cyclohexenone compound havingthe structure:

wherein each of X and Y independently is oxygen, NR₅ or sulfur;

-   -   R is a hydrogen or C(═O)C₁-C₈alkyl;    -   each of R₁, R₂ and R₃ independently is a hydrogen, methyl or        (CH₂)_(m)—CH₃;    -   R₄ is NR₅R₆, OR₅, OC(═O)R₇, C(═O)OR₅, C(═O)R₅, C(═O)NR₅R₆,        halogen, 5 or 6-membered lactone, C₁-C₈alkyl, C₂-C₈alkenyl,        C₂-C₈alkynyl, aryl, glucosyl, wherein the 5 or 6-membered        lactone, C₁-C₈alkyl, C₂-C₈alkenyl, C₂-C₈alkynyl, aryl, and        glucosyl are optionally substituted with one or more        substituents selected from NR₅R₆, OR₅, OC(═O)R₇, C(═O)OR₅,        C(═O)R₅, C(═O)NR₅R₆, C₁-C₈ alkyl, C₂-C₈ alkenyl, C₂-C₈ alkynyl,        C₃-C₈ cycloalkyl, and C₁-C₈ haloalkyl;    -   each of R₅ and R₆ is independently a hydrogen or C₁-C₈alkyl;    -   R₇ is a C₁-C₈alkyl, OR₅ or NR₅R₆;    -   m=1-12; and    -   n=1-12; or a pharmaceutically acceptable salt, metabolite,        solvate or prodrug thereof.

In another aspect provided herein are methods for the treatment of aβ-Amyloid induced disease in a subject, comprising administering to thesubject affected by a disease resulting from hyperglycemia in need atherapeutically effective amount of a cyclohexenone compound having thestructure:

wherein each of X and Y independently is oxygen, NR₅ or sulfur;

-   -   R is a hydrogen or C(═O)C₁-C₈alkyl;    -   each of R₁, R₂ and R₃ independently is a hydrogen, methyl or        (CH₂)_(m)—CH₃;    -   R₄ is NR₅R₆, OR₅, OC(═O)R₇, C(═O)OR₅, C(═O)R₅, C(═O)NR₅R₆,        halogen, 5 or 6-membered lactone, C₁-C₈alkyl, C₂-C₈alkenyl,        C₂-C₈alkynyl, aryl, glucosyl, wherein the 5 or 6-membered        lactone, C₁-C₈alkyl, C₂-C₈alkenyl, C₂-C₈alkynyl, aryl, and        glucosyl are optionally substituted with one or more        substituents selected from NR₅R₆, OR₅, OC(═O)R₇, C(═O)OR₅,        C(═O)R₅, C(═O)NR₅R₆, C₁-C₈ alkyl, C₂-C₈ alkenyl, C₂-C₈ alkynyl,        C₃-C₈ cycloalkyl, and C₁-C₈ haloalkyl;    -   each of R₅ and R₆ is independently a hydrogen or C₁-C₈alkyl;    -   R₇ is a C₁-C₈alkyl, OR₅ or NR₅R₆;    -   m=1-12; and    -   n=1-12; or a pharmaceutically acceptable salt, metabolite,        solvate or prodrug thereof.

In another aspect provided herein are methods for treating Alzheimer'sdisease in a subject, comprising administering to the subject affectedby a disease resulting from hyperglycemia in need a therapeuticallyeffective amount of a cyclohexenone compound having the structure:

wherein each of X and Y independently is oxygen, NR₅ or sulfur;

-   -   R is a hydrogen or C(═O)C₁-C₈alkyl;    -   each of R₁, R₂ and R₃ independently is a hydrogen, methyl or        (CH₂)_(m)—CH₃;    -   R₄ is NR₅R₆, OR₅, OC(═O)R₇, C(═O)OR₅, C(═O)R₅, C(═O)NR₅R₆,        halogen, 5 or 6-membered lactone, C₁-C₈alkyl, C₂-C₈alkenyl,        C₂-C₈alkynyl, aryl, glucosyl, wherein the 5 or 6-membered        lactone, C₁-C₈alkyl, C₂-C₈alkenyl, C₂-C₈alkynyl, aryl, and        glucosyl are optionally substituted with one or more        substituents selected from NR₅R₆, OR₅, OC(═O)R₇, C(═O)OR₅,        C(═O)R₅, C(═O)NR₅R₆, C₁-C₈ alkyl, C₂-C₈ alkenyl, C₂-C₈ alkynyl,        C₃-C₈ cycloalkyl, and C₁-C₈ haloalkyl;    -   each of R₅ and R₆ is independently a hydrogen or C₁-C₈alkyl;    -   R₇ is a C₁-C₈alkyl, OR₅ or NR₅R₆;    -   m=1-12; and    -   n=1-12; or a pharmaceutically acceptable salt, metabolite,        solvate or prodrug thereof.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIG. 1 shows viability of PC12 cells treated with exemplary Compound 1.Cells were cultured under 10% serum (A) or serum-free (B) conditions andtreated with different concentrations of Compound 1 as indicated for 2days. Cell viability was detected using Cell Counting Kit-8 (CCK-8). Theerror bars represented mean±SEM from two independent experiments.

FIG. 2A-D show illustrative effective results of the exemplarycyclohexenone compound such as Compound 1 on cell viability induced byβ-amyloid. PC12 cells were cultured under 10% serum (A)(B) or serum-free(C)(D) conditions and pretreated with Compound 1 (A)(C) or β-amyloid(B)(D) as indicated for 24 h. Cell viability was detected using CCK-8.The error bars represented mean±SEM from three independentexperiments. * indicated P<0.05, ** P<0.01, and ***P<0.001 compared withthe Aβ-treated control.

DETAILED DESCRIPTION OF THE INVENTION

The process of neurodegeneration is not well understood so the diseasesthat stem from it have, as yet, no cures. Several experimentaltherapeutic agents are in clinical trials. In some embodiments, providedherein are methods for the treatment of neurodegenerative diseases byadministering a cyclohexenone compound described herein to a subject(e.g. a human). The cyclohexenone compounds provide therapeutic benefitto a subject being treated for neurodegenerative diseases (see Examples1-5). The cyclohexenone compounds, in some embodiments, are obtainedfrom extracts of natural products and provide reduced complicationsand/or side effects. In some embodiments, this invention provides thetherapeutic and prophylactic potential of exemplary cyclohexenonecompounds (e.g., Compound 1) for treating or reducing the risk ofneurodegenerative diseases such as Alzheimer's disease, Parkinson'sdisease, multiple sclerosis, stroke syndromes, amyotrophic lateralsclerosis, or the like.

The term “neurodegenerative disease (or disorder)” as used herein refersto an abnormality in a mammal in which neuronal integrity is threatened.Neuronal integrity can be threatened when neuronal cells displaydecreased survival or when the neurons can no longer propagate a signal.Examples of neurodegenerative processes include stroke syndromes,subarachnoid hemorrhage, brain dysfunction post-brain surgery, disordersof the nervous system due to hypoxia, hypoglycemia, brain or spinaldamage, intoxication with drugs or gases, administration ofchemotherapy, alcohol and the like and examples of neurodegenerativediseases include Alzheimer's disease, amyotrophic lateral sclerosis,Parkinson's disease, myasthenia gravis, HIV-related encephalitis,cervical spondylosis, multiple sclerosis, Down's syndrome, andHuntington's chorea. A key to curing these diseases is control ofneuronal death including apoptosis. The cyclohexenone compounds of thisinvention may be administered systemically to one who is afflicted withneurodegenerative diseases or to patients who are believed to besusceptible to such diseases.

Cellular models composed of primary neuronal cultures or neuron-likecell lines are commonly used to study neuron cell death and to test theneuroprotective properties of specific compounds. Cellular models areeasily accessible, permitting dissection and modulation of signalingpathways involved in neuron death. Due to the relative difficulty ofstudying signaling in neurons, neurotrophin signaling has been primarilystudied using the pheochromocytoma PC12 cells as a model system. Thiscell line has proved useful for studying mechanisms of neuronalsurvival, differentiation, and cell death. For example, they synthesize,store, and release two common neurotransmitters: dopamine andnorepinephrine. PC12 cells release these neurotransmitters in responseto potassium ions, acetylcholine, or nicotine, just as a neuron would.Furthermore, PC12 cells exhibit two growth patterns. When they are grownas undifferentiated cells, they divide approximately every 24-48 hours.Second, when PC12 cells are exposed to nerve growth factor (NGF), theyexhibit a different growth pattern. Initially, they cease cellulardivision. Afterwards, they flatten out and develop long projectionsknown as neurites. The neurites often grow to be in close contact with aneighboring cell, resembling a neuronal synapse. If the nerve growthfactor is removed, the PC12 cells will eventually return to theirroughly spherical shape and resume cell division (Greene et al., (1999)Methodologies for the Culture and Experimental Use of the PC12 RatPheochromocytoma Cell Line. In: Culturing Nerve Cells G. Banker, K.Goslin eds., MIT Press, Cambridge, Mass. pp 161-187). NGF withdrawalsimilarly triggers death of sympathetic neurons both in vivo and invitro.

To optimize the exemplary Compound 1 concentration for NGF-induced PC12neural model, titration experiments were performed to determine the IC₅₀values of Compound 1 in PC12. IC₅₀ values of the exemplary inventionCompound 1 are determined to be 57.98 mg/ml (in 10% serum supplementedmedia) and 13.39 mg/ml (serum-free media) respectively by cell viabilityassay (FIG. 1).

To assess the prophylactic potential of the invention cyclohexenonecompound (i.e., Compound 1) the NGF-induced PC12 cells were pretreatedwith different concentrations of Compound 1 (25 and 50 mg/ml in 10%serum-supplemented medium; 0.3 and 3 mg/ml in serum-free medium) andthen challenged with 10 μM β-Amyloid (Aβ) before determination of cellviability. The cell viability results indicate that the exemplaryCompound 1 exhibits a significant impact in preventing (reducing therisk of) cells from Aβ-induced neuronal damage in either serum orserum-free culture conditions (FIGS. 2A and 2C). The similar experimentwas conducted to assess the therapeutic effect of Compound 1. PC12 cellswere pretreated with 10 μM Aβ for 24 h in serum and serum-free mediumseparately first and then challenged with different concentrations ofCompound 1 before determination of the cell viability. The results showthat the treatment with low-dose Compound 1 dramatically inhibits thedamage induced by Aβ and improves the cell viability (FIGS. 2B and 2D).

Clearly, the exemplary cyclohexenone compounds, such as Compound 1,exhibit therapeutic and prophylactic effectiveness in NGF-induced PC12neural model. Thus, the invention cyclohexenone compounds providedherein such as Compound 1 are useful for preventing or treatingneurodegenerative disease such as AD.

In some embodiments, there are provided methods for treating or reducingthe risk of neurodegenerative disease comprising administering to asubject a therapeutically effective amount of a cyclohexenone compoundhaving the structure:

wherein each of X and Y independently is oxygen, NR₅ or sulfur;

-   -   R is a hydrogen or C(═O)C₁-C₈alkyl;    -   each of R₁, R₂ and R₃ independently is a hydrogen, methyl or        (CH₂)_(m)—CH₃;    -   R₄ is NR₅R₆, OR₅, OC(═O)R₇, C(═O)OR₅, C(═O)R₅, C(═O)NR₅R₆,        halogen, 5 or 6-membered lactone, C₁-C₈alkyl, C₂-C₈alkenyl,        C₂-C₈alkynyl, aryl, glucosyl, wherein the 5 or 6-membered        lactone, C₁-C₈alkyl, C₂-C₈alkenyl, C₂-C₈alkynyl, aryl, and        glucosyl are optionally substituted with one or more        substituents selected from NR₅R₆, OR₅, OC(═O)R₇, C(═O)OR₅,        C(═O)R₅, C(═O)NR₅R₆, C₁-C₈ alkyl, C₂-C₈ alkenyl, C₂-C₈ alkynyl,        C₃-C₈ cycloalkyl, and C₁-C₈ haloalkyl;    -   each of R₅ and R₆ is independently a hydrogen or C₁-C₈alkyl;    -   R₇ is a C₁-C₈alkyl, OR₅ or NR₅R₆;    -   m=1-12; and    -   n=1-12; or a pharmaceutically acceptable salt, metabolite,        solvate or prodrug thereof.

In some embodiments, the methods inhibit β-Amyloid induced neuronaldamage in a subject. In some embodiments, the neurodegenerative diseaseis selected from the group consisting of Alzheimer's disease,Parkinson's disease, multiple sclerosis, stroke syndromes, andamyotrophic lateral sclerosis. In certain embodiments, theneurodegenerative disease is Alzheimer's disease. In some embodiments,the cyclohexenone compound inhibits β-Amyloid induced neuronal damage ina subject. In some embodiments, the subject is human. See Examples 2-5.

In some embodiments, the cyclohexenone compound having the structure

is prepared synthetically or semi-synthetically from any suitablestarting material. In other embodiments, the cyclohexenone compound isprepared by fermentation, or the like. For example, Compound 1 (alsoknown as Antroquinonol™ or “Antroq”) or Compound 3, in some instances,is prepared from 4-hydroxy-2,3-dimethoxy-6-methylcyclohexa-2,5-dienone.The non-limited exemplary compounds are illustrated below.

In other embodiments, the cyclohexenone compound having the structure

is isolated from the organic solvent extracts of Antrodia camphorata. Insome embodiments, the organic solvent is selected from alcohols (e.g.,methanol, ethanol, propanol, or the like), esters (e.g., methyl acetate,ethyl acetate, or the like), alkanes (e.g., pentane, hexane, heptane, orthe like), halogenated alkanes (e.g., chloromethane, chloroethane,chloroform, methylene chloride, and the like), and the like. Forexample, exemplary Compounds 1-7 are isolated from organic solventextracts. In certain embodiments, the organic solvent is alcohol. Incertain embodiments, the alcohol is ethanol. In some embodiments, thecyclohexenone compound is isolated from the aqueous extracts of Antrodiacamphorata.

In some embodiments, R is a hydrogen, C(═O)C₃H₈, C(═O)C₂H₅, or C(═O)CH₃.In some embodiments, R₁ is a hydrogen or methyl. In certain embodiments,R₂ is a hydrogen, methyl, ethyl, propyl, butyl, pentyl or hexyl. In someembodiments, R₃ is a hydrogen, methyl, ethyl, propyl, butyl, pentyl orhexyl. In some embodiments, R₄ is halogen, NH₂, NHCH₃, N(CH₃)₂, OCH₃,OC₂H₅, C(═O)CH₃, C(═O)C₂H₅, C(═O)OCH₃, C(═O)OC₂H₅, C(═O)NHCH₃,C(═O)NHC₂H₅, C(═O)NH₂, OC(═O)CH₃, OC(═O)C₂H₅, OC(═O)OCH₃, OC(═O)OC₂H₅,OC(═O)NHCH₃, OC(═O)NHC₂H₅, or OC(═O)NH₂. In some embodiments, R₄ isC₂H₅C(CH₃)₂OH, C₂H₅C(CH₃)₂OCH₃, CH₂COOH, C₂H₅COOH, CH₂OH, C₂H₅OH, CH₂Ph,C₂H₅Ph, CH₂CH═C(CH₃)(CHO), CH₂CH═C(CH₃)(C(═O)CH₃), 5 or 6-memberedlactone, C₁-C₈ alkyl, C₂-C₈alkenyl, C₂-C₈alkynyl, aryl, and glucosyl,wherein the 5 or 6-membered lactone, C₁-C₈ alkyl, C₂-C₈alkenyl,C₂-C₈alkynyl, aryl, and glucosyl are optionally substituted with one ormore substituents selected from NR₅R₆, OR₅, OC(═O)R₇, C(═O)OR₅, C(═O)R₅,C(═O)NR₅R₆, C₁-C₈ alkyl, C₂-C₈ alkenyl, C₂-C₈ alkynyl, C₃-C₈ cycloalkyl,and C₁-C₈ haloalkyl. In certain embodiments, R₄ is CH₂CH═C(CH₃)₂. Incertain embodiments, the compound is

In some embodiments, there are provided methods treating a β-Amyloidinduced disease in a subject, comprising administering to the subjectaffected by a disease resulting from hyperglycemia in need atherapeutically effective amount of a cyclohexenone compound having thestructure:

wherein each of X and Y independently is oxygen, NR₅ or sulfur;

-   -   R is a hydrogen or C(═O)C₁-C₈alkyl;    -   each of R₁, R₂ and R₃ independently is a hydrogen, methyl or        (CH₂)_(m)—CH₃;    -   R₄ is NR₅R₆, OR₅, OC(═O)R₇, C(═O)OR₅, C(═O)R₅, C(═O)NR₅R₆,        halogen, 5 or 6-membered lactone, C₁-C₈alkyl, C₂-C₈alkenyl,        C₂-C₈alkynyl, aryl, glucosyl, wherein the 5 or 6-membered        lactone, C₁-C₈alkyl, C₂-C₈alkenyl, C₂-C₈alkynyl, aryl, and        glucosyl are optionally substituted with one or more        substituents selected from NR₅R₆, OR₅, OC(═O)R₇, C(═O)OR₅,        C(═O)R₅, C(═O)NR₅R₆, C₁-C₈ alkyl, C₂-C₈ alkenyl, C₂-C₈ alkynyl,        C₃-C₈ cycloalkyl, and C₁-C₈ haloalkyl;    -   each of R₅ and R₆ is independently a hydrogen or C₁-C₈alkyl;    -   R₇ is a C₁-C₈alkyl, OR₅ or NR₅R₆;    -   m=1-12; and    -   n=1-12; or a pharmaceutically acceptable salt, metabolite,        solvate or prodrug thereof.

In some embodiments, the β-Amyloid induced disease is Alzheimer'sdisease or Down's syndrome. In certain embodiments, the subject ishuman.

Certain Pharmaceutical and Medical Terminology

Unless otherwise stated, the following terms used in this application,including the specification and claims, have the definitions givenbelow. It must be noted that, as used in the specification and theappended claims, the singular forms “a,” “an” and “the” include pluralreferents unless the context clearly dictates otherwise. Unlessotherwise indicated, conventional methods of mass spectroscopy, NMR,HPLC, protein chemistry, biochemistry, recombinant DNA techniques andpharmacology are employed. In this application, the use of “or” or “and”means “and/or” unless stated otherwise. Furthermore, use of the term“including” as well as other forms, such as “include”, “includes,” and“included,” is not limiting. The section headings used herein are fororganizational purposes only and are not to be construed as limiting thesubject matter described.

An “alkyl” group refers to an aliphatic hydrocarbon group. The alkylgroup may be a saturated alkyl group (which means that it does notcontain any carbon-carbon double bonds or carbon-carbon triple bonds) orthe alkyl group may be an unsaturated alkyl group (which means that itcontains at least one carbon-carbon double bonds or carbon-carbon triplebond). The alkyl moiety, whether saturated or unsaturated, may bebranched, or straight chain.

The “alkyl” group may have 1 to 12 carbon atoms (whenever it appearsherein, a numerical range such as “1 to 12 refers to each integer in thegiven range; e.g., “1 to 12 carbon atoms” means that the alkyl group mayconsist of 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc., up toand including 12 carbon atoms, although the present definition alsocovers the occurrence of the term “alkyl” where no numerical range isdesignated). The alkyl group of the compounds described herein may bedesignated as “C₁-C₈ alkyl” or similar designations. By way of exampleonly, “C₁-C₈ alkyl” indicates that there are one, two, three, four,five, six, seven or eight carbon atoms in the alkyl chain. In one aspectthe alkyl is selected from the group consisting of methyl, ethyl,propyl, iso-propyl, n-butyl, iso-butyl, sec-butyl, and t-butyl. Typicalalkyl groups include, but are in no way limited to, methyl, ethyl,propyl, isopropyl, butyl, isobutyl, sec-butyl, tertiary butyl, pentyl,neopentyl, hexyl, allyl, but-2-enyl, but-3-enyl, cyclopropylmethyl,cyclobutylmethyl, cyclopentylmethyl, cyclohexylmethyl, and the like. Inone aspect, an alkyl is a C₁-C₈ alkyl.

The term “alkylene” refers to a divalent alkyl radical. Any of the abovementioned monovalent alkyl groups may be an alkylene by abstraction of asecond hydrogen atom from the alkyl. In one aspect, an alkylene is aC₁-C₁₂alkylene. In another aspect, an alkylene is a C₁-C₈alkylene.Typical alkylene groups include, but are not limited to, —CH₂—,—CH(CH₃)—, —C(CH₃)₂—, —CH₂CH₂—, —CH₂CH(CH₃)—, —CH₂C(CH₃)₂—, —CH₂CH₂CH₂—,—CH₂CH₂CH₂CH₂—, and the like.

As used herein, the term “aryl” refers to an aromatic ring wherein eachof the atoms forming the ring is a carbon atom. Aryl rings are formed byfive, six, seven, eight, nine, or more than nine carbon atoms. Arylgroups are optionally substituted. In one aspect, an aryl is a phenyl ora naphthalenyl. In one aspect, an aryl is a phenyl. In one aspect, anaryl is a C₆-C₁₀aryl. Depending on the structure, an aryl group can be amonoradical or a diradical (i.e., an arylene group). In one aspect, anarylene is a C₆-C₁₀ arylene. Exemplary arylenes include, but are notlimited to, phenyl-1,2-ene, phenyl-1,3-ene, and phenyl-1,4-ene.

The term “aromatic” refers to a planar ring having a delocalizedπ-electron system containing 4n+2 π electrons, where n is an integer.Aromatic rings can be formed from five, six, seven, eight, nine, ten, ormore than ten atoms. Aromatics are optionally substituted. The term“aromatic” includes both carbocyclic aryl (“aryl”, e.g., phenyl) andheterocyclic aryl (or “heteroaryl” or “heteroaromatic”) groups (e.g.,pyridine). The term includes monocyclic or fused-ring polycyclic (i.e.,rings which share adjacent pairs of carbon atoms) groups.

The term “halo” or, alternatively, “halogen” or “halide” means fluoro,chloro, bromo or iodo.

The term “lactone” refers to a cyclic ester which can be seen as thecondensation product of an alcohol group —OH and a carboxylic acid group—COOH in the same molecule. It is characterized by a closed ringconsisting of two or more carbon atoms and a single oxygen atom, with aketone group ═O in one of the carbons adjacent to the other oxygen.

The term “heterocycle” or “heterocyclic” refers to heteroaromatic rings(also known as heteroaryls) and heterocycloalkyl rings (also known asheteroalicyclic groups) containing one to four heteroatoms in thering(s), where each heteroatom in the ring(s) is selected from O, S andN, wherein each heterocyclic group has from 4 to 10 atoms in its ringsystem, and with the proviso that the any ring does not contain twoadjacent O or S atoms. Non-aromatic heterocyclic groups (also known asheterocycloalkyls) include groups having only 3 atoms in their ringsystem, but aromatic heterocyclic groups must have at least 5 atoms intheir ring system. The heterocyclic groups include benzo-fused ringsystems. An example of a 3-membered heterocyclic group is aziridinyl. Anexample of a 4-membered heterocyclic group is azetidinyl. An example ofa 5-membered heterocyclic group is thiazolyl. An example of a 6-memberedheterocyclic group is pyridyl, and an example of a 10-memberedheterocyclic group is quinolinyl. Examples of non-aromatic heterocyclicgroups are pyrrolidinyl, tetrahydrofuranyl, dihydrofuranyl,tetrahydrothienyl, oxazolidinonyl, tetrahydropyranyl, dihydropyranyl,tetrahydrothiopyranyl, piperidinyl, morpholinyl, thiomorpholinyl,thioxanyl, piperazinyl, aziridinyl, azetidinyl, oxetanyl, thietanyl,homopiperidinyl, oxepanyl, thiepanyl, oxazepinyl, diazepinyl,thiazepinyl, 1,2,3,6-tetrahydropyridinyl, pyrrolin-2-yl, pyrrolin-3-yl,indolinyl, 2H-pyranyl, 4H-pyranyl, dioxanyl, 1,3-dioxolanyl,pyrazolinyl, dithianyl, dithiolanyl, dihydropyranyl, dihydrothienyl,dihydrofuranyl, pyrazolidinyl, imidazolinyl, imidazolidinyl,3-azabicyclo[3.1.0]hexanyl, 3-azabicyclo[4.1.0]heptanyl, 3H-indolyl andquinolizinyl. Examples of aromatic heterocyclic groups are pyridinyl,imidazolyl, pyrimidinyl, pyrazolyl, triazolyl, pyrazinyl, tetrazolyl,furyl, thienyl, isoxazolyl, thiazolyl, oxazolyl, isothiazolyl, pyrrolyl,quinolinyl, isoquinolinyl, indolyl, benzimidazolyl, benzofuranyl,cinnolinyl, indazolyl, indolizinyl, phthalazinyl, pyridazinyl,triazinyl, isoindolyl, pteridinyl, purinyl, oxadiazolyl, thiadiazolyl,furazanyl, benzofurazanyl, benzothiophenyl, benzothiazolyl,benzoxazolyl, quinazolinyl, quinoxalinyl, naphthyridinyl, andfuropyridinyl. The foregoing groups may be C-attached or N-attachedwhere such is possible. For instance, a group derived from pyrrole maybe pyrrol-1-yl (N-attached) or pyrrol-3-yl (C-attached). Further, agroup derived from imidazole may be imidazol-1-yl or imidazol-3-yl (bothN-attached) or imidazol-2-yl, imidazol-4-yl or imidazol-5-yl (allC-attached). The heterocyclic groups include benzo-fused ring systems.Non-aromatic heterocycles may be substituted with one or two oxo (═O)moieties, such as pyrrolidin-2-one.

The term “alkenyl” as used herein, means a straight, branched chain, orcyclic (in which case, it would also be known as a “cycloalkenyl”)hydrocarbon containing from 2-10 carbons and containing at least onecarbon-carbon double bond formed by the removal of two hydrogens. Insome embodiments, depending on the structure, an alkenyl group is amonoradical or a diradical (i.e., an alkenylene group). In someembodiments, alkenyl groups are optionally substituted. Illustrativeexamples of alkenyl include, but are not limited to, ethenyl,2-propenyl, 2-methyl-2-propenyl, 3-butenyl, 4-pentenyl, 5-hexenyl,2-heptenyl, 2-methyl-1-heptenyl, and 3-cecenyl.

The term “alkynyl” as used herein, means a straight, branched chain, orcyclic (in which case, it would also be known as a “cycloalkenyl”)hydrocarbon containing from 2-10 carbons and containing at least onecarbon-carbon triple bond formed by the removal of four hydrogens. Insome embodiments, depending on the structure, an alkynyl group is amonoradical or a diradical (i.e., an alkynylene group). In someembodiments, alkynyl groups are optionally substituted. Illustrativeexamples of alkynyl include, but are not limited to, ethynyl, propynyl,butynyl, pentynyl, hexynyl, heptynyl, and the like.

The term “alkoxy” as used herein, means an alkyl group, as definedherein, appended to the parent molecular moiety through an oxygen atom.Illustrative examples of alkoxy include, but are not limited to,methoxy, ethoxy, propoxy, 2-propoxy, butoxy, tert-butoxy, pentyloxy, andhexyloxy.

The term “cycloalkyl” as used herein, means a monocyclic or polycyclicradical that contains only carbon and hydrogen, and includes those thatare saturated, partially unsaturated, or fully unsaturated. Cycloalkylgroups include groups having from 3 to 10 ring atoms. Representativeexamples of cyclic include but are not limited to, the followingmoieties:

In some embodiments, depending on the structure, a cycloalkyl group is amonoradical or a diradical (e.g., a cycloalkylene group).

The terms “haloalkyl,” “haloalkenyl,” “haloalkynyl” and “haloalkoxy” asused herein, include alkyl, alkenyl, alkynyl and alkoxy structures inwhich at least one hydrogen is replaced with a halogen atom. In certainembodiments in which two or more hydrogen atoms are replaced withhalogen atoms, the halogen atoms are all the same as one another. Inother embodiments in which two or more hydrogen atoms are replaced withhalogen atoms, the halogen atoms are not all the same as one another.The terms “fluoroalkyl” and “fluoroalkoxy” include haloalkyl andhaloalkoxy groups, respectively, in which the halo is fluorine. Incertain embodiments, haloalkyls are optionally substituted.

The term “glucosyl” as used herein, include D- or L-form glucosylgroups, in which the glucosyl group is attached via any hydroxyl groupon the glucose ring.

The term “acceptable” with respect to a formulation, composition oringredient, as used herein, means having no persistent detrimentaleffect on the general health of the subject being treated.

Antrodia is a genus of fungi in the family Meripilaceae. Antrodiaspecies have fruiting bodies that typically lie flat or spread out onthe growing surface, with the hymenium exposed to the outside; the edgesmay be turned so as to form narrow brackets. Most species are found intemperate and boreal forests, and cause brown rot.

The term “carrier,” as used herein, refers to relatively nontoxicchemical compounds or agents that facilitate the incorporation of acompound into cells or tissues.

The terms “co-administration” or the like, as used herein, are meant toencompass administration of the selected therapeutic agents to a singlepatient, and are intended to include treatment regimens in which theagents are administered by the same or different route of administrationor at the same or different time.

The term “diluent” refers to chemical compounds that are used to dilutethe compound of interest prior to delivery. Diluents can also be used tostabilize compounds because they can provide a more stable environment.Salts dissolved in buffered solutions (which also can provide pH controlor maintenance) are utilized as diluents in the art, including, but notlimited to a phosphate buffered saline solution.

The terms “effective amount” or “therapeutically effective amount,” asused herein, refer to a sufficient amount of an agent or a compoundbeing administered which will relieve to some extent one or more of thesymptoms of the disease or condition being treated. The result can bereduction and/or alleviation of the signs, symptoms, or causes of adisease, or any other desired alteration of a biological system. Forexample, an “effective amount” for therapeutic uses is the amount of thecomposition comprising a compound as disclosed herein required toprovide a clinically significant decrease in disease symptoms. Anappropriate “effective” amount in any individual case may be determinedusing techniques, such as a dose escalation study.

The terms “enhance” or “enhancing,” as used herein, means to increase orprolong either in potency or duration a desired effect. Thus, in regardto enhancing the effect of therapeutic agents, the term “enhancing”refers to the ability to increase or prolong, either in potency orduration, the effect of other therapeutic agents on a system. An“enhancing-effective amount,” as used herein, refers to an amountadequate to enhance the effect of another therapeutic agent in a desiredsystem.

A “metabolite” of a compound disclosed herein is a derivative of thatcompound that is formed when the compound is metabolized. The term“active metabolite” refers to a biologically active derivative of acompound that is formed when the compound is metabolized. The term“metabolized,” as used herein, refers to the sum of the processes(including, but not limited to, hydrolysis reactions and reactionscatalyzed by enzymes) by which a particular substance is changed by anorganism. Thus, enzymes may produce specific structural alterations to acompound. For example, cytochrome P450 catalyzes a variety of oxidativeand reductive reactions while uridine diphosphate glucuronyltransferasescatalyze the transfer of an activated glucuronic-acid molecule toaromatic alcohols, aliphatic alcohols, carboxylic acids, amines and freesulphydryl groups. Metabolites of the compounds disclosed herein areoptionally identified either by administration of compounds to a hostand analysis of tissue samples from the host, or by incubation ofcompounds with hepatic cells in vitro and analysis of the resultingcompounds.

The term “pharmaceutical combination” as used herein, means a productthat results from the mixing or combining of more than one activeingredient and includes both fixed and non-fixed combinations of theactive ingredients. The term “fixed combination” means that the activeingredients, e.g. a compound (i.e., a cyclohexenone compound describedherein) and a co-agent, are both administered to a patientsimultaneously in the form of a single entity or dosage. The term“non-fixed combination” means that the active ingredients, e.g. acompound (i.e., a cyclohexenone compound described herein) and aco-agent, are administered to a patient as separate entities eithersimultaneously, concurrently or sequentially with no specificintervening time limits, wherein such administration provides effectivelevels of the two compounds in the body of the patient. The latter alsoapplies to cocktail therapy, e.g. the administration of three or moreactive ingredients.

The term “pharmaceutical composition” refers to a mixture of a compound(i.e., a cyclohexenone compound described herein) with other chemicalcomponents, such as carriers, stabilizers, diluents, dispersing agents,suspending agents, thickening agents, and/or excipients. Thepharmaceutical composition facilitates administration of the compound toan organism. Multiple techniques of administering a compound exist inthe art including, but not limited to: intravenous, oral, aerosol,parenteral, ophthalmic, pulmonary and topical administration.

The term “subject” or “patient” encompasses mammals. Examples of mammalsinclude, but are not limited to, any member of the Mammalian class:humans, non-human primates such as chimpanzees, and other apes andmonkey species; farm animals such as cattle, horses, sheep, goats,swine; domestic animals such as rabbits, dogs, and cats; laboratoryanimals including rodents, such as rats, mice and guinea pigs, and thelike. In one embodiment, the mammal is a human.

The terms “treat,” “treating” or “treatment,” as used herein, includealleviating, abating or ameliorating at least one symptom of a diseaseor condition, preventing additional symptoms, inhibiting the disease orcondition, e.g., arresting the development of the disease or condition,relieving the disease or condition, causing regression of the disease orcondition, relieving a condition caused by the disease or condition, orstopping the symptoms of the disease or condition eitherprophylactically and/or therapeutically.

Routes of Administration and Dosage

Suitable routes of administration include, but are not limited to, oral,intravenous, rectal, aerosol, parenteral, ophthalmic, pulmonary,transmucosal, transdermal, vaginal, otic, nasal, and topicaladministration. In addition, by way of example only, parenteral deliveryincludes intramuscular, subcutaneous, intravenous, intramedullaryinjections, as well as intrathecal, direct intraventricular,intraperitoneal, intralymphatic, and intranasal injections.

In certain embodiments, a compound as described herein is administeredin a local rather than systemic manner, for example, via injection ofthe compound directly into an organ, often in a depot preparation orsustained release formulation. In specific embodiments, long actingformulations are administered by implantation (for examplesubcutaneously or intramuscularly) or by intramuscular injection.Furthermore, in other embodiments, the drug is delivered in a targeteddrug delivery system, for example, in a liposome coated withorgan-specific antibody. In such embodiments, the liposomes are targetedto and taken up selectively by the organ. In yet other embodiments, thecompound as described herein is provided in the form of a rapid releaseformulation, in the form of an extended release formulation, or in theform of an intermediate release formulation. In yet other embodiments,the compound described herein is administered topically.

In some embodiments, the cyclohexenone compound, or a pharmaceuticallyacceptable salt, metabolite, solvate or prodrug thereof, is administeredparenterally or intravenously. In other embodiments, the cyclohexenonecompound, or a pharmaceutically acceptable salt, metabolite, solvate orprodrug thereof, is administered by injection. In some embodiments, thecyclohexenone compound, or a pharmaceutically acceptable salt,metabolite, solvate or prodrug thereof, is administered orally.

In the case wherein the patient's condition does not improve, upon thedoctor's discretion the administration of the compounds may beadministered chronically, that is, for an extended period of time,including throughout the duration of the patient's life in order toameliorate or otherwise control or limit the symptoms of the patient'sdisease or condition. In the case wherein the patient's status doesimprove, upon the doctor's discretion the administration of thecompounds may be given continuously or temporarily suspended for acertain length of time (i.e., a “drug holiday”).

The foregoing ranges are merely suggestive, as the number of variablesin regard to an individual treatment regime is large, and considerableexcursions from these recommended values are not uncommon. Such dosagesmay be altered depending on a number of variables, not limited to theactivity of the compound used, the disease or condition to be treated,the mode of administration, the requirements of the individual subject,the severity of the disease or condition being treated, and the judgmentof the practitioner.

Toxicity and therapeutic efficacy of such therapeutic regimens can bedetermined by standard pharmaceutical procedures in cell cultures orexperimental animals, including, but not limited to, for determining theLD₅₀ (the dose lethal to 50% of the population) and the ED₅₀ (the dosetherapeutically effective in 50% of the population). The dose ratiobetween the toxic and therapeutic effects is the therapeutic index andit can be expressed as the ratio between LD₅₀ and ED₅₀. Compoundsexhibiting high therapeutic indices are preferred. The data obtainedfrom cell culture assays and animal studies can be used in formulating arange of dosage for use in human. The dosage of such compounds liespreferably within a range of circulating concentrations that include theED₅₀ with minimal toxicity. The dosage may vary within this rangedepending upon the dosage form employed and the route of administrationutilized.

Pharmaceutical Formulation

In some embodiments provide pharmaceutical compositions comprising atherapeutically effective amount of a cyclohexenone compound having thestructure:

-   -   wherein each of X and Y independently is oxygen, NR₅ or sulfur;    -   R is a hydrogen or C(═O)C₁-C₈alkyl;    -   each of R₁, R₂ and R₃ independently is a hydrogen, methyl or        (CH₂)_(m)—CH₃;    -   R₄ is NR₅R₆, OR₅, OC(═O)R₇, C(═O)OR₅, C(═O)R₅, C(═O)NR₅R₆,        halogen, 5 or 6-membered lactone, C₁-C₈alkyl, C₂-C₈alkenyl,        C₂-C₈alkynyl, aryl, glucosyl, wherein the 5 or 6-membered        lactone, C₁-C₈alkyl, C₂-C₈alkenyl, C₂-C₈alkynyl, aryl, and        glucosyl are optionally substituted with one or more        substituents selected from NR₅R₆, OR₅, OC(═O)R₇, C(═O)OR₅,        C(═O)R₅, C(═O)NR₅R₆, C₁-C₈ alkyl, C₂-C₈ alkenyl, C₂-C₈ alkynyl,        C₃-C₈ cycloalkyl, and C₁-C₈ haloalkyl;    -   each of R₅ and R₆ is independently a hydrogen or C₁-C₈alkyl;    -   R₇ is a C₁-C₈alkyl, OR₅ or NR₅R₆;    -   m=1-12; and n=1-12; or a pharmaceutically acceptable salt,        metabolite, solvate or prodrug thereof; and a pharmaceutically        acceptable excipient.

In some embodiments, the cyclohexenone compounds of the pharmaceuticalcompositions have the structure:

-   -   wherein each of X and Y independently is oxygen, NR₅ or sulfur;    -   R is a hydrogen or C(═O)C₁-C₈alkyl;    -   each of R₁, R₂ and R₃ independently is a hydrogen, methyl or        (CH₂)_(m)—CH₃;    -   R₄ is NR₅R₆, OR₅, OC(═O)R₇, C(═O)OR₅, C(═O)R₅, C(═O)NR₅R₆,        halogen, 5 or 6-membered lactone, C₁-C₈alkyl, C₂-C₈alkenyl,        C₂-C₈alkynyl, aryl, glucosyl, wherein the 5 or 6-membered        lactone, C₁-C₈alkyl, C₂-C₈alkenyl, C₂-C₈alkynyl, aryl, and        glucosyl are optionally substituted with one or more        substituents selected from NR₅R₆, OR₅, OC(═O)R₇, C(═O)OR₅,        C(═O)R₅, C(═O)NR₅R₆, C₁-C₈ alkyl, C₂-C₈ alkenyl, C₂-C₈ alkynyl,        C₃-C₈ cycloalkyl, and C₁-C₈ haloalkyl;    -   each of R₅ and R₆ is independently a hydrogen or C₁-C₈alkyl;    -   R₇ is a C₁-C₈alkyl, OR₅ or NR₅R₆;    -   m=1-12; and n=1-12; or a pharmaceutically acceptable salt,        metabolite, solvate or prodrug thereof.

In some embodiments, R is a hydrogen, C(═O)C₃H₈, C(═O)C₂H₅, or C(═O)CH₃.In some embodiments, each of R₁, R₂ and R₃ independently is a hydrogen,methyl, ethyl, propyl, butyl, pentyl hexyl, heptyl, or octyl. In certainembodiments, R₁ is a hydrogen or methyl. In certain embodiments, R₂ is ahydrogen, methyl, ethyl, propyl, butyl, pentyl or hexyl. In certainembodiments, R₃ is a hydrogen, methyl, ethyl, propyl, butyl, pentyl orhexyl. In some embodiments, R₄ is halogen, NH₂, NHCH₃, N(CH₃)₂, OCH₃,OC₂H₅, C(═O)CH₃, C(═O)C₂H₅, C(═O)OCH₃, C(═O)OC₂H₅, C(═O)NHCH₃,C(═O)NHC₂H₅, C(═O)NH₂, OC(═O)CH₃, OC(═O)C₂H₅, OC(═O)OCH₃, OC(═O)OC₂H₅,OC(═O)NHCH₃, OC(═O)NHC₂H₅, or OC(═O)NH₂. In certain embodiments, R₄ isC₂H₅C(CH₃)₂OH, C₂H₅C(CH₃)₂OCH₃, CH₂COOH, C₂H₅COOH, CH₂OH, C₂H₅OH, CH₂Ph,C₂H₅Ph, CH₂CH═C(CH₃)(CHO), CH₂CH═C(CH₃)(C(═O)CH₃), 5 or 6-memberedlactone, aryl, or glucosyl, wherein the 5 or 6-membered lactone, aryl,and glucosyl are optionally substituted with one or more substituentsselected from NR₅R₆, OR₅, OC(═O)R₇, C(═O)OR₅, C(═O)R₅, C(═O)NR₅R₆, C₁-C₈alkyl, C₂-C₈ alkenyl, C₂-C₈ alkynyl, C₃-C₈ cycloalkyl, and C₁-C₈haloalkyl. In certain embodiments, R₄ is CH₂COOH, C₂H₅COOH, CH₂OH,C₂H₅OH, CH₂Ph, C₂H₅Ph, CH₂CH═C(CH₃)(CHO), CH₂CH═C(CH₃)(C(═O)CH₃), C₁-C₈alkyl, 5 or 6-membered lactone, aryl, or glucosyl, wherein the 5 or6-membered lactone, C₁-C₈ alkyl, aryl, and glucosyl are optionallysubstituted with one or more substituents selected from NR₅R₆, OR₅,OC(═O)R₇, C(═O)OR₅, C(═O)R₅, C(═O)NR₅R₆, C₁-C₈ alkyl, C₂-C₈ alkenyl,C₂-C₈ alkynyl, C₃-C₈ cycloalkyl, and C₁-C₈ haloalkyl.

In certain embodiments, the compound is selected from group consistingof

In certain embodiments, the compound is selected from group consistingof

In some embodiments, the compounds described herein are formulated intopharmaceutical compositions. In specific embodiments, pharmaceuticalcompositions are formulated in a conventional manner using one or morephysiologically acceptable carriers comprising excipients andauxiliaries which facilitate processing of the active compounds intopreparations which can be used pharmaceutically. Proper formulation isdependent upon the route of administration chosen. Any pharmaceuticallyacceptable techniques, carriers, and excipients are used as suitable toformulate the pharmaceutical compositions described herein: Remington:The Science and Practice of Pharmacy, Nineteenth Ed (Easton, Pa.: MackPublishing Company, 1995); Hoover, John E., Remington's PharmaceuticalSciences, Mack Publishing Co., Easton, Pa. 1975; Liberman, H. A. andLachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York,N.Y., 1980; and Pharmaceutical Dosage Forms and Drug Delivery Systems,Seventh Ed. (Lippincott Williams & Wilkins 1999).

Provided herein are pharmaceutical compositions comprising a compound(i.e., a cyclohexenone compound described herein) and a pharmaceuticallyacceptable diluent(s), excipient(s), or carrier(s). In certainembodiments, the compounds described are administered as pharmaceuticalcompositions in which a compound (i.e., a cyclohexenone compounddescribed herein) is mixed with other active ingredients, as incombination therapy. Encompassed herein are all combinations of activesset forth in the combination therapies section below and throughout thisdisclosure. In specific embodiments, the pharmaceutical compositionsinclude one or more compounds (i.e., a cyclohexenone compound describedherein).

A pharmaceutical composition, as used herein, refers to a mixture of acompound (i.e., a cyclohexenone compound described herein) with otherchemical components, such as carriers, stabilizers, diluents, dispersingagents, suspending agents, thickening agents, and/or excipients. Incertain embodiments, the pharmaceutical composition facilitatesadministration of the compound to an organism. In some embodiments,practicing the methods of treatment or use provided herein,therapeutically effective amounts of compounds (i.e., a cyclohexenonecompound described herein) are administered in a pharmaceuticalcomposition to a mammal having a disease or condition to be treated. Inspecific embodiments, the mammal is a human. In certain embodiments,therapeutically effective amounts vary depending on the severity of thedisease, the age and relative health of the subject, the potency of thecompound used and other factors. The compounds described herein are usedsingly or in combination with one or more therapeutic agents ascomponents of mixtures.

In one embodiment, a compound (i.e., a cyclohexenone compound describedherein) is formulated in an aqueous solution. In specific embodiments,the aqueous solution is selected from, by way of example only, aphysiologically compatible buffer, such as Hank's solution, Ringer'ssolution, or physiological saline buffer. In other embodiments, acompound (i.e., a cyclohexenone compound described herein) is formulatedfor transmucosal administration. In specific embodiments, transmucosalformulations include penetrants that are appropriate to the barrier tobe permeated. In still other embodiments wherein the compounds describedherein are formulated for other parenteral injections, appropriateformulations include aqueous or nonaqueous solutions. In specificembodiments, such solutions include physiologically compatible buffersand/or excipients.

In another embodiment, compounds described herein are formulated fororal administration. Compounds described herein, including a compound(i.e., a cyclohexenone compound described herein), are formulated bycombining the active compounds with, e.g., pharmaceutically acceptablecarriers or excipients. In various embodiments, the compounds describedherein are formulated in oral dosage forms that include, by way ofexample only, tablets, powders, pills, dragees, capsules, liquids, gels,syrups, elixirs, slurries, suspensions and the like.

In certain embodiments, pharmaceutical preparations for oral use areobtained by mixing one or more solid excipients with one or more of thecompounds described herein, optionally grinding the resulting mixture,and processing the mixture of granules, after adding suitableauxiliaries, if desired, to obtain tablets or dragee cores. Suitableexcipients are, in particular, fillers such as sugars, includinglactose, sucrose, mannitol, or sorbitol; cellulose preparations such as:for example, maize starch, wheat starch, rice starch, potato starch,gelatin, gum tragacanth, methylcellulose, microcrystalline cellulose,hydroxypropylmethylcellulose, sodium carboxymethylcellulose; or otherssuch as: polyvinylpyrrolidone (PVP or povidone) or calcium phosphate. Inspecific embodiments, disintegrating agents are optionally added.Disintegrating agents include, by way of example only, cross-linkedcroscarmellose sodium, polyvinylpyrrolidone, agar, or alginic acid or asalt thereof such as sodium alginate.

In one embodiment, dosage forms, such as dragee cores and tablets, areprovided with one or more suitable coating. In specific embodiments,concentrated sugar solutions are used for coating the dosage form. Thesugar solutions, optionally contain additional components, such as byway of example only, gum arabic, talc, polyvinylpyrrolidone, carbopolgel, polyethylene glycol, and/or titanium dioxide, lacquer solutions,and suitable organic solvents or solvent mixtures. Dyestuffs and/orpigments are also optionally added to the coatings for identificationpurposes. Additionally, the dyestuffs and/or pigments are optionallyutilized to characterize different combinations of active compounddoses.

In certain embodiments, therapeutically effective amounts of at leastone of the compounds described herein are formulated into other oraldosage forms. Oral dosage forms include push-fit capsules made ofgelatin, as well as soft, sealed capsules made of gelatin and aplasticizer, such as glycerol or sorbitol. In specific embodiments,push-fit capsules contain the active ingredients in admixture with oneor more filler. Fillers include, by way of example only, lactose,binders such as starches, and/or lubricants such as talc or magnesiumstearate and, optionally, stabilizers. In other embodiments, softcapsules, contain one or more active compound that is dissolved orsuspended in a suitable liquid. Suitable liquids include, by way ofexample only, one or more fatty oil, liquid paraffin, or liquidpolyethylene glycol. In addition, stabilizers are optionally added.

In other embodiments, therapeutically effective amounts of at least oneof the compounds described herein are formulated for buccal orsublingual administration. Formulations suitable for buccal orsublingual administration include, by way of example only, tablets,lozenges, or gels. In still other embodiments, the compounds describedherein are formulated for parental injection, including formulationssuitable for bolus injection or continuous infusion. In specificembodiments, formulations for injection are presented in unit dosageform (e.g., in ampoules) or in multi-dose containers. Preservatives are,optionally, added to the injection formulations. In still otherembodiments, the pharmaceutical compositions of a compound (i.e., acyclohexenone compound described herein) are formulated in a formsuitable for parenteral injection as a sterile suspensions, solutions oremulsions in oily or aqueous vehicles. Parenteral injection formulationsoptionally contain formulatory agents such as suspending, stabilizingand/or dispersing agents. In specific embodiments, pharmaceuticalformulations for parenteral administration include aqueous solutions ofthe active compounds in water-soluble form. In additional embodiments,suspensions of the active compounds are prepared as appropriate oilyinjection suspensions. Suitable lipophilic solvents or vehicles for usein the pharmaceutical compositions described herein include, by way ofexample only, fatty oils such as sesame oil, or synthetic fatty acidesters, such as ethyl oleate or triglycerides, or liposomes. In certainspecific embodiments, aqueous injection suspensions contain substanceswhich increase the viscosity of the suspension, such as sodiumcarboxymethyl cellulose, sorbitol, or dextran. Optionally, thesuspension contains suitable stabilizers or agents which increase thesolubility of the compounds to allow for the preparation of highlyconcentrated solutions. Alternatively, in other embodiments, the activeingredient is in powder form for constitution with a suitable vehicle,e.g., sterile pyrogen-free water, before use.

In one aspect, compounds (i.e., cyclohexenone compounds describedherein) are prepared as solutions for parenteral injection as describedherein or known in the art and administered with an automatic injector.Automatic injectors, such as those disclosed in U.S. Pat. Nos.4,031,893, 5,358,489; 5,540,664; 5,665,071, 5,695,472 and WO/2005/087297(each of which are incorporated herein by reference for such disclosure)are known. In general, all automatic injectors contain a volume ofsolution that includes a compound (i.e., a cyclohexenone compounddescribed herein) to be injected. In general, automatic injectorsinclude a reservoir for holding the solution, which is in fluidcommunication with a needle for delivering the drug, as well as amechanism for automatically deploying the needle, inserting the needleinto the patient and delivering the dose into the patient. Exemplaryinjectors provide about 0.3 mL, 0.6 mL, 1.0 mL or other suitable volumeof solution at about a concentration of 0.5 mg to 50 mg of a compound(i.e., a cyclohexenone compound described herein) per 1 mL of solution.Each injector is capable of delivering only one dose of the compound.

In still other embodiments, the compounds (i.e., cyclohexenone compoundsdescribed herein) are administered topically. The compounds describedherein are formulated into a variety of topically administrablecompositions, such as solutions, suspensions, lotions, gels, pastes,medicated sticks, balms, creams or ointments. Such pharmaceuticalcompositions optionally contain solubilizers, stabilizers, tonicityenhancing agents, buffers and preservatives.

In yet other embodiments, the compounds (i.e., cyclohexenone compoundsdescribed herein) are formulated for transdermal administration. Inspecific embodiments, transdermal formulations employ transdermaldelivery devices and transdermal delivery patches and can be lipophilicemulsions or buffered, aqueous solutions, dissolved and/or dispersed ina polymer or an adhesive. In various embodiments, such patches areconstructed for continuous, pulsatile, or on demand delivery ofpharmaceutical agents. In additional embodiments, the transdermaldelivery of a compound (i.e., a cyclohexenone compound described herein)is accomplished by means of iontophoretic patches and the like. Incertain embodiments, transdermal patches provide controlled delivery ofa compound (i.e., a cyclohexenone compound described herein). Inspecific embodiments, the rate of absorption is slowed by usingrate-controlling membranes or by trapping the compound within a polymermatrix or gel. In alternative embodiments, absorption enhancers are usedto increase absorption. Absorption enhancers or carriers includeabsorbable pharmaceutically acceptable solvents that assist passagethrough the skin. For example, in one embodiment, transdermal devicesare in the form of a bandage comprising a backing member, a reservoircontaining the compound optionally with carriers, optionally a ratecontrolling barrier to deliver the compound to the skin of the host at acontrolled and predetermined rate over a prolonged period of time, andmeans to secure the device to the skin.

Transdermal formulations described herein may be administered using avariety of devices which have been described in the art. For example,such devices include, but are not limited to, U.S. Pat. Nos. 3,598,122,3,598,123, 3,710,795, 3,731,683, 3,742,951, 3,814,097, 3,921,636,3,972,995, 3,993,072, 3,993,073, 3,996,934, 4,031,894, 4,060,084,4,069,307, 4,077,407, 4,201,211, 4,230,105, 4,292,299, 4,292,303,5,336,168, 5,665,378, 5,837,280, 5,869,090, 6,923,983, 6,929,801 and6,946,144.

The transdermal dosage forms described herein may incorporate certainpharmaceutically acceptable excipients which are conventional in theart. In one embodiment, the transdermal formulations described hereininclude at least three components: (1) a formulation of a compound(i.e., a cyclohexenone compound described herein); (2) a penetrationenhancer; and (3) an aqueous adjuvant. In addition, transdermalformulations can include additional components such as, but not limitedto, gelling agents, creams and ointment bases, and the like. In someembodiments, the transdermal formulations further include a woven ornon-woven backing material to enhance absorption and prevent the removalof the transdermal formulation from the skin. In other embodiments, thetransdermal formulations described herein maintain a saturated orsupersaturated state to promote diffusion into the skin.

In other embodiments, the compounds (i.e., cyclohexenone compoundsdescribed herein) are formulated for administration by inhalation.Various forms suitable for administration by inhalation include, but arenot limited to, aerosols, mists or powders. Pharmaceutical compositionsof a compound (i.e., a cyclohexenone compound described herein) areconveniently delivered in the form of an aerosol spray presentation frompressurized packs or a nebuliser, with the use of a suitable propellant(e.g., dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide or other suitable gas). Inspecific embodiments, the dosage unit of a pressurized aerosol isdetermined by providing a valve to deliver a metered amount. In certainembodiments, capsules and cartridges of, such as, by way of exampleonly, gelatins for use in an inhaler or insufflator are formulatedcontaining a powder mix of the compound and a suitable powder base suchas lactose or starch.

Intranasal formulations are known in the art and are described in, forexample, U.S. Pat. Nos. 4,476,116, 5,116,817 and 6,391,452, each ofwhich is specifically incorporated herein by reference. Formulations,which include a compound (i.e., a cyclohexenone compound describedherein), which are prepared according to these and other techniqueswell-known in the art are prepared as solutions in saline, employingbenzyl alcohol or other suitable preservatives, fluorocarbons, and/orother solubilizing or dispersing agents known in the art. See, forexample, Ansel, H. C. et al., Pharmaceutical Dosage Forms and DrugDelivery Systems, Sixth Ed. (1995). Preferably these compositions andformulations are prepared with suitable nontoxic pharmaceuticallyacceptable ingredients. These ingredients are found in sources such asREMINGTON: THE SCIENCE AND PRACTICE OF PHARMACY, 21st edition, 2005, astandard reference in the field. The choice of suitable carriers ishighly dependent upon the exact nature of the nasal dosage form desired,e.g., solutions, suspensions, ointments, or gels. Nasal dosage formsgenerally contain large amounts of water in addition to the activeingredient. Minor amounts of other ingredients such as pH adjusters,emulsifiers or dispersing agents, preservatives, surfactants, gellingagents, or buffering and other stabilizing and solubilizing agents mayalso be present. Preferably, the nasal dosage form should be isotonicwith nasal secretions.

For administration by inhalation, the compounds described herein, may bein a form as an aerosol, a mist or a powder. Pharmaceutical compositionsdescribed herein are conveniently delivered in the form of an aerosolspray presentation from pressurized packs or a nebuliser, with the useof a suitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol, the dosageunit may be determined by providing a valve to deliver a metered amount.Capsules and cartridges of, such as, by way of example only, gelatin foruse in an inhaler or insufflator may be formulated containing a powdermix of the compound described herein and a suitable powder base such aslactose or starch.

In still other embodiments, the compounds (i.e., cyclohexenone compoundsdescribed herein) are formulated in rectal compositions such as enemas,rectal gels, rectal foams, rectal aerosols, suppositories, jellysuppositories, or retention enemas, containing conventional suppositorybases such as cocoa butter or other glycerides, as well as syntheticpolymers such as polyvinylpyrrolidone, PEG, and the like. In suppositoryforms of the compositions, a low-melting wax such as, but not limitedto, a mixture of fatty acid glycerides, optionally in combination withcocoa butter is first melted.

In certain embodiments, pharmaceutical compositions are formulated inany conventional manner using one or more physiologically acceptablecarriers comprising excipients and auxiliaries which facilitateprocessing of the active compounds into preparations which can be usedpharmaceutically. Proper formulation is dependent upon the route ofadministration chosen. Any pharmaceutically acceptable techniques,carriers, and excipients is optionally used as suitable and asunderstood in the art. Pharmaceutical compositions comprising a compound(i.e., a cyclohexenone compound described herein) may be manufactured ina conventional manner, such as, by way of example only, by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping or compression processes.

Pharmaceutical compositions include at least one pharmaceuticallyacceptable carrier, diluent or excipient and at least one compound(i.e., cyclohexenone compounds described herein) described herein as anactive ingredient. The active ingredient is in free-acid or free-baseform, or in a pharmaceutically acceptable salt form. In addition, themethods and pharmaceutical compositions described herein include the usecrystalline forms (also known as polymorphs), as well as activemetabolites of these compounds having the same type of activity. Alltautomers of the compounds described herein are included within thescope of the compounds presented herein. Additionally, the compoundsdescribed herein encompass unsolvated as well as solvated forms withpharmaceutically acceptable solvents such as water, ethanol, and thelike. The solvated forms of the compounds presented herein are alsoconsidered to be disclosed herein. In addition, the pharmaceuticalcompositions optionally include other medicinal or pharmaceuticalagents, carriers, adjuvants, such as preserving, stabilizing, wetting oremulsifying agents, solution promoters, salts for regulating the osmoticpressure, buffers, and/or other therapeutically valuable substances.

Methods for the preparation of compositions comprising the compoundsdescribed herein include formulating the compounds with one or moreinert, pharmaceutically acceptable excipients or carriers to form asolid, semi-solid or liquid. Solid compositions include, but are notlimited to, powders, tablets, dispersible granules, capsules, cachets,and suppositories. Liquid compositions include solutions in which acompound is dissolved, emulsions comprising a compound, or a solutioncontaining liposomes, micelles, or nanoparticles comprising a compoundas disclosed herein. Semi-solid compositions include, but are notlimited to, gels, suspensions and creams. The form of the pharmaceuticalcompositions described herein include liquid solutions or suspensions,solid forms suitable for solution or suspension in a liquid prior touse, or as emulsions. These compositions also optionally contain minoramounts of nontoxic, auxiliary substances, such as wetting oremulsifying agents, pH buffering agents, and so forth.

In some embodiments, pharmaceutical composition comprising at leastcompound (i.e., cyclohexenone compounds described herein) illustrativelytakes the form of a liquid where the agents are present in solution, insuspension or both. Typically when the composition is administered as asolution or suspension a first portion of the agent is present insolution and a second portion of the agent is present in particulateform, in suspension in a liquid matrix. In some embodiments, a liquidcomposition includes a gel formulation. In other embodiments, the liquidcomposition is aqueous.

In certain embodiments, pharmaceutical aqueous suspensions include oneor more polymers as suspending agents. Polymers include water-solublepolymers such as cellulosic polymers, e.g., hydroxypropylmethylcellulose, and water-insoluble polymers such as cross-linkedcarboxyl-containing polymers. Certain pharmaceutical compositionsdescribed herein include a mucoadhesive polymer, selected from, forexample, carboxymethylcellulose, carbomer (acrylic acid polymer),poly(methylmethacrylate), polyacrylamide, polycarbophil, acrylicacid/butyl acrylate copolymer, sodium alginate and dextran.

Pharmaceutical compositions also, optionally include solubilizing agentsto aid in the solubility of a compound (i.e., cyclohexenone compoundsdescribed herein). The term “solubilizing agent” generally includesagents that result in formation of a micellar solution or a truesolution of the agent. Certain acceptable nonionic surfactants, forexample polysorbate 80, are useful as solubilizing agents, as canophthalmically acceptable glycols, polyglycols, e.g., polyethyleneglycol 400, and glycol ethers.

Furthermore, pharmaceutical compositions optionally include one or morepH adjusting agents or buffering agents, including acids such as acetic,boric, citric, lactic, phosphoric and hydrochloric acids; bases such assodium hydroxide, sodium phosphate, sodium borate, sodium citrate,sodium acetate, sodium lactate and tris-hydroxymethylaminomethane; andbuffers such as citrate/dextrose, sodium bicarbonate and ammoniumchloride. Such acids, bases and buffers are included in an amountrequired to maintain pH of the composition in an acceptable range.

Additionally, pharmaceutical compositions optionally include one or moresalts in an amount required to bring osmolality of the composition intoan acceptable range. Such salts include those having sodium, potassiumor ammonium cations and chloride, citrate, ascorbate, borate, phosphate,bicarbonate, sulfate, thiosulfate or bisulfite anions; suitable saltsinclude sodium chloride, potassium chloride, sodium thiosulfate, sodiumbisulfite and ammonium sulfate.

Other pharmaceutical compositions optionally include one or morepreservatives to inhibit microbial activity. Suitable preservativesinclude mercury-containing substances such as merfen and thiomersal;stabilized chlorine dioxide; and quaternary ammonium compounds such asbenzalkonium chloride, cetyltrimethylammonium bromide andcetylpyridinium chloride.

Still other pharmaceutical compositions include one or more surfactantsto enhance physical stability or for other purposes. Suitable nonionicsurfactants include polyoxyethylene fatty acid glycerides and vegetableoils, e.g., polyoxyethylene (60) hydrogenated castor oil; andpolyoxyethylene alkylethers and alkylphenyl ethers, e.g., octoxynol 10,octoxynol 40.

Still other pharmaceutical compositions may include one or moreantioxidants to enhance chemical stability where required. Suitableantioxidants include, by way of example only, ascorbic acid and sodiummetabisulfite.

In certain embodiments, pharmaceutical aqueous suspension compositionsare packaged in single-dose non-reclosable containers. Alternatively,multiple-dose reclosable containers are used, in which case it istypical to include a preservative in the composition.

In alternative embodiments, other delivery systems for hydrophobicpharmaceutical compounds are employed. Liposomes and emulsions areexamples of delivery vehicles or carriers herein. In certainembodiments, organic solvents such as N-methylpyrrolidone are alsoemployed. In additional embodiments, the compounds described herein aredelivered using a sustained-release system, such as semipermeablematrices of solid hydrophobic polymers containing the therapeutic agent.Various sustained-release materials are useful herein. In someembodiments, sustained-release capsules release the compounds for a fewhours up to over 24 hours. Depending on the chemical nature and thebiological stability of the therapeutic reagent, additional strategiesfor protein stabilization may be employed.

In certain embodiments, the formulations described herein include one ormore antioxidants, metal chelating agents, thiol containing compoundsand/or other general stabilizing agents. Examples of such stabilizingagents, include, but are not limited to: (a) about 0.5% to about 2% w/vglycerol, (b) about 0.1% to about 1% w/v methionine, (c) about 0.1% toabout 2% w/v monothioglycerol, (d) about 1 mM to about 10 mM EDTA, (e)about 0.01% to about 2% w/v ascorbic acid, (f) 0.003% to about 0.02% w/vpolysorbate 80, (g) 0.001% to about 0.05% w/v. polysorbate 20, (h)arginine, (i) heparin, (j) dextran sulfate, (k) cyclodextrins, (l)pentosan polysulfate and other heparinoids, (m) divalent cations such asmagnesium and zinc; or (n) combinations thereof.

Combination Treatments

In general, the compositions described herein and, in embodiments wherecombinational therapy is employed, other agents do not have to beadministered in the same pharmaceutical composition, and in someembodiments, because of different physical and chemical characteristics,are administered by different routes. In some embodiments, the initialadministration is made according to established protocols, and then,based upon the observed effects, the dosage, modes of administration andtimes of administration is modified by the skilled clinician.

In some embodiments, therapeutically-effective dosages vary when thedrugs are used in treatment combinations. Combination treatment furtherincludes periodic treatments that start and stop at various times toassist with the clinical management of the patient. For combinationtherapies described herein, dosages of the co-administered compoundsvary depending on the type of co-drug employed, on the specific drugemployed, on the disease, disorder, or condition being treated and soforth.

It is understood that in some embodiments, the dosage regimen to treat,prevent, or ameliorate the condition(s) for which relief is sought, ismodified in accordance with a variety of factors. These factors includethe disorder from which the subject suffers, as well as the age, weight,sex, diet, and medical condition of the subject. Thus, in otherembodiments, the dosage regimen actually employed varies widely andtherefore deviates from the dosage regimens set forth herein.

Combinations of compounds (i.e., the cyclohexenone compound describedherein) with other neurodegenerative disease therapeutic agents areintended to be covered. In some embodiments, examples ofneurodegenerative disease such as AD therapeutic agents include, but arenot limited to, the following: donepezil (Aricept®), Galantamine(RAZADYNE™, Reminyl®), rivastigmine (Exelon®), tacrine (Cognex®),memantine (Namenda®), and the like.

The combinations of the cyclohexenone compounds and otherneurodegenerative disease therapeutic agents described herein encompassadditional therapies and treatment regimens with other agents in someembodiments. Such additional therapies and treatment regimens caninclude another neurodegenerative disease therapy in some embodiments.Alternatively, in other embodiments, additional therapies and treatmentregimens include other agents used to treat adjunct conditionsassociated with the neurodegenerative disease or a side effect from suchagent in the combination therapy. In further embodiments, adjuvants orenhancers are administered with a combination therapy described herein.

In some embodiments provide compositions for treating or reducing therisk of neurodegenerative diseases comprising a therapeuticallyeffective amount of a cyclohexenone compound having the structure:

-   -   wherein each of X and Y independently is oxygen, NR₅ or sulfur;    -   R is a hydrogen or C(═O)C₁-C₈alkyl;    -   each of R₁, R₂ and R₃ independently is a hydrogen, methyl or        (CH₂)_(m)—CH₃;    -   R₄ is NR₅R₆, OR₅, OC(═O)R₇, C(═O)OR₅, C(═O)R₅, C(═O)NR₅R₆,        halogen, 5 or 6-membered lactone, C₁-C₈alkyl, C₂-C₈alkenyl,        C₂-C₈alkynyl, aryl, glucosyl, wherein the 5 or 6-membered        lactone, C₁-C₈alkyl, C₂-C₈alkenyl, C₂-C₈alkynyl, aryl, and        glucosyl are optionally substituted with one or more        substituents selected from NR₅R₆, OR₅, OC(═O)R₇, C(═O)OR₅,        C(═O)R₅, C(═O)NR₅R₆, C₁-C₈ alkyl, C₂-C₈ alkenyl, C₂-C₈ alkynyl,        C₃-C₈ cycloalkyl, and C₁-C₈ haloalkyl;    -   each of R₅ and R₆ is independently a hydrogen or C₁-C₈alkyl;    -   R₇ is a C₁-C₈alkyl, OR₅ or NR₅R₆;    -   m=1-12; and n=1-12; or a pharmaceutically acceptable salt,        metabolite, solvate or prodrug thereof; and one or more        neurodegenerative disease therapeutic agents.

EXAMPLES Example 1. Preparation of the Exemplary Cyclohexenone Compounds

One hundred grams of mycelia, fruiting bodies or mixture of both fromAntrodia camphorata were placed into a flask. A proper amount of waterand alcohol (70-100% alcohol solution) was added into the flask and werestirred at 20-25° C. for at least 1 hour. The solution was filteredthrough a filter and 0.45 μm membrane and the filtrate was collected asthe extract.

The filtrate of Antrodia camphorata was subjected to High PerformanceLiquid chromatography (HPLC) analysis. The separation was performed on aRP18 column, the mobile phase consisted of methanol (A) and 0.3% aceticacid (B), with the gradient conditions of 0-10 min in 95%-20% B, 10-20min in 20%-10% B, 20-35 min in 10%-10% B, 35-40 min in 10%-95% B, at theflow rate of 1 ml/min. The column effluent was monitored with aUV-visible detector.

The fractions collected at 21.2 to 21.4 min were collected andconcentrated to yield compound 5, a product of pale yellow liquid.Compound 5 was analyzed to be4-hydroxy-5-(11-hydroxy-3,7,11-trimethyldodeca-2,6-dienyl)-2,3-dimethoxy-6-methylcyclohex-2-enonewith molecular weight of 408 (Molecular formula: C₂₄H₄₀O₅). ¹H-NMR(CDCl₃) δ (ppm)=1.21, 1.36, 1.67, 1.71, 1.75, 1.94, 2.03, 2.07, 2.22,2.25, 3.68, 4.05, 5.71 and 5.56. ¹³C-NMR (CDCl₃) δ(ppm): 12.31, 16.1,16.12, 17.67, 25.67, 26.44, 26.74, 27.00, 30.10, 40.27, 43.34, 59.22,60.59, 71.8, 120.97, 123.84, 124.30, 131.32, 134.61, 135.92, 138.05,160.45, and 197.11.

Compound 5:4-hydroxy-5-(11-hydroxy-3,7,11-trimethyldodeca-2,6-dienyl)-2,3-dimethoxy-6-methylcyclohex-2-enone

The fractions collected at 23.7 to 24.0 min were collected andconcentrated to yield compound 7, a product of pale yellow liquid.Compound 7 was analyzed to be4-hydroxy-2,3-dimethoxy-5-(11-methoxy-3,7,11-trimethyldodeca-2,6-dienyl)-6-methylcyclohex-2-enonewith molecular weight of 422 (C₂₅H₄₂O₅). ¹H-NMR (CDCl₃) δ (ppm)=1.21,1.36, 1.71, 1.75, 1.94, 2.03, 2.07, 2.22, 2.25, 3.24, 3.68, 4.05, 5.12,5.50, and 5.61. ¹³C-NMR (CDCl₃) δ(ppm): 12.31, 16.1, 16.12, 17.67,24.44, 26.44, 26.74, 27.00, 37.81, 39.81, 40.27, 43.34, 49.00, 59.22,60.59, 120.97, 123.84, 124.30, 135.92, 138.05, 160.45 and 197.12.

Compound 7:4-hydroxy-2,3-dimethoxy-5-(11-methoxy-3,7,11-trimethyldodeca-2,6-dienyl)-6-methylcyclohex-2-enone

The fractions collected at 25 to 30 min were collected and concentratedto yield4-hydroxy-2,3-dimethoxy-6-methyl-5-(3,7,11-trimethyldodeca-2,6,10-trienyl)cyclohex-2-enone(compound 1), a product of pale yellow brown liquid. The analysis ofcompound 1 showed the molecular formula of C₂₄H₃₈O₄, molecular weight of390 with melting point of 48 to 52° C. NMR spectra showed that ¹H-NMR(CDCl₃) δ (ppm)=1.51, 1.67, 1.71, 1.75, 1.94, 2.03, 2.07, 2.22, 2.25,3.68, 4.05, 5.07, and 5.14; ¹³C-NMR (CDCl₃) δ (ppm)=12.31, 16.1, 16.12,17.67, 25.67, 26.44, 26.74, 27.00, 39.71, 39.81, 40.27, 43.34, 59.22,60.59, 120.97, 123.84, 124.30, 131.32, 135.35, 135.92, 138.05, 160.45,and 197.12.

Compound 1:4-hydroxy-2,3-dimethoxy-6-methyl-5-(3,7,11-trimethyldodeca-2,6,10-trienyl)cyclohex-2-enone

Compound 6, a metabolite of compound 1, was obtained from urine samplesof rats fed with Compound 1 in the animal study. Compound 6 wasdetermined to be 4-hydroxy-2,3-dimethoxy-6-methyl-5-(3-methyl-2-hexenoicacid)cyclohex-2-enone with molecular weight of 312 (C₁₆H₂₄O₆). Compound4 which was determined as3,4-dihydroxy-2-methoxy-6-methyl-5-(3,7,11-trimethyldodeca-2,6,10-trienyl)cyclohex-2-enone(molecular weight of 376, C₂₃H₃₆O₄), was obtained when compound 1 wasunder the condition of above 40° C. for 6 hours.

Alternatively, the exemplary compounds may be prepared from4-hydroxy-2,3-dimethoxy-6-methylcyclohexa-2,5-dienone, or the like.Similarly, other cyclohexenone compounds having the structure

are isolated from Antrodia camphorata or prepared synthetically orsemi-synthetically from the suitable starting materials. An ordinaryskilled in the art would readily utilize appropriate conditions for suchsynthesis.

Example 2. Preparation and Maintenance of PC12 Cultures

PC12 is a cell line derived from a pheochromocytoma of the rat adrenalmedulla. PC12 cells stop dividing and terminally differentiate whentreated with nerve growth factor. This makes PC12 cells, and the like,useful as a model system for neuronal differentiation.

Rat pheochromocytoma PC12 cells, purchased from Bioresource Collectionand Research Center (BCRC), were cultured in RPMI1640 medium (Gibco, MD)containing 10% fetal calf serum, 5% horse donor serum, 100 μg/mlstreptomycin, 100 units/ml penicillin, 2 mM L-glutamine, and maintainedin humidified incubator at 37° C. with 5% CO₂. Cells were transferredtwice a week. Experiments were carried out in 96-well plates coated withpoly-_(D)-lysine (1 mg/mL) to assist cell adhesion. PC12 cells weregrown on medium with mouse submaxillary-gland nerve growth factor (NGF2.5S, 100 ng/ml; Invitrogen, Carlsbad, Calif.) for 48 h to inducedifferentiation into neuron-like cells before all experiments conductedin this study.

Example 3. Experimental Treatments of Compound 1 and β-Amyloid on PC12Cultures

To optimize the exemplary Compound 1 concentration for NGF-induced PC12neural model, titration experiments were performed to determine the IC₅₀values of Compound 1 in PC12. PC12 Cells (1×10⁴ cells) were incubatedwith various concentrations of Compound 1 (0.001, 0.01, 0.1, 1, 10, 100μg/ml in a 10% serum-supplemented medium; 0.3, 1, 3, 10, 30, 100 μg/mlin serum-free medium) for 48 h.

β-Amyloid₁₋₄₀ (Aβ) (Invitrogen, Carlsbad, Calif.) was dissolved in 0.1%(v/v) trifluoroacetic acid (TFA) in water at 10 mg/ml and stored at −20°C. as a stock solution. Aβ was diluted to 0.5 mg/ml withphosphate-buffered saline solution (PBS, without Ca2+) and aggregate at25° C. for 48 h before use. To investigate the therapeutic effect ofexemplary Compound 1 on Alzheimer disease (AD) cell model in vitro,cultures were pretreated with Aβ for 24 h and then exposed to freshlyprepared Compound 1 for 24 h before cell viability measurements. Incontrast, to investigate the prophylactic effect of Compound 1 on ADcell model, cultures were pretreated with Compound 1 for 24 h and thenexposure to aggregated Aβ for 24 h before cell viability measurements.

Example 4: CCK-8 Cell Viability Assay

Cell Counting Kit-8 (CCK-8) allows sensitive colorimetric assays for thedetermination of cell viability in cell proliferation and cytotoxicityassays. The detection sensitivity of CCK-8 is higher than the othertetrazolium salts such as MTT, XTT, MTS or WST-1.

The viability of cells was measured using the Cell Counting Kit-8(CCK-8, Enzo, Life Sciences). A highly water-soluble tetrazolium salt,WST-8, is reduced by dehydrogenases in cells to give a yellow-coloredproduct (formazan), which is soluble in the culture medium. The amountof the fomazan dye generated by the activity of dehydrogenases in cellsis directly proportional to the number of living cells. Aftertreatments, the CCK-8 solution was added to each well of plates andincubates the plate for 4 h in the incubator. The concentration of theformazan product was determined spectrophotometrically at an absorbancewavelength 450 nm and cell viability was expressed as a percentage ofthe corresponding control.

IC₅₀ values of the exemplary invention Compound 1 are determined to be57.98 μg/ml (in 10% serum supplemented media) and 13.39 mg/ml(serum-free media) respectively by cell viability assay (FIG. 1). Toassess the prophylactic potential of the invention cyclohexenonecompound (i.e., Compound 1) the NGF-induced PC12 cells were pretreatedwith different concentrations of Compound 1 (25 and 50 mg/ml in 10%serum-supplemented medium; 0.3 and 3 μg/ml in serum-free medium) for 24h. After washing, the cells were then challenged with 10 μM β-Amyloid(Aβ) for another 24 h before determination of cell viability. The cellviability results indicate that the exemplary Compound 1 exhibits asignificant impact in preventing (reducing the risk of) cells fromAβ-induced neuronal damage in either serum or serum-free cultureconditions (FIGS. 2A and 2C). The similar experiment was conducted toassess the therapeutic effect of Compound 1. PC12 cells were pretreatedwith 10 μM Aβ for 24 h in serum and serum-free medium separately first.After washing, PC 12 cells were then challenged with differentconcentrations of Compound 1 as indicated for another 24 h beforedetermination of the cell viability. The results showed that thetreatment with low-dose Compound 1 dramatically inhibited the damageinduced by Aβ and improved the cell viability (FIGS. 2B and 2D).

Example 5: Animal Study of Parkinson's Disease with Compound 1

Thirty adult Wistar rats are randomly divided into five groups namelycontrol, 6-OHDA model, and Compound 1 (25, 50, and 100 mg/kg body weightsuspended in one ml of 0.1% carboxymethyl cellulose). The treatment isstarted three days before surgery and continued for next 14 days. Thesurgery is done on third day in all groups for administration of 6-OHDAinto the right striatum and right substantia nigra, whereas controlgroup injects with 6-OHDA vehicle. Various behavior and biochemicaltests (Apomorphine-induced rotational behavior, Stepping test,Initiation time, Postural balance test, and Disengage time) are used toevaluate the neuroprotective effect of Compound 1. One-way analysis ofvariance (ANOVA) followed by Dunnett's test is used to compareinter-group differences. P<0.05 is considered as statisticallysignificant.

Animals:

Thirty male adult Wistar rats, weighing 200 to 250 g, are maintainedunder 12: 12-h light: dark cycle with food and water provided adlibitum.

Experimental Design:

The animals are randomly allocated to five groups as Vehicle, 6-OHDA,Compound 1 (25, 50, and 100 mg/kg body weight) of six rats each. Vehicleand 6-OHDA groups are received 1 ml of 0.1% carboxymethyl cellulose(CMC) solution in water while Compound 1 groups are treated with theselected doses, suspended in 1 ml of 0.1% CMC in water. All treatmentsare given orally; daily in morning at 10 am, for 17 days (includes threedays pre-treatment before surgery). On the third day, brain surgery isperformed, after 60 minutes of each treatment, in all groups for 6-OHDAadministration except vehicle-treated group where vehicle isadministered.

Operative Procedure:

Unilateral striatal lesion is produced by stereotaxic injection of 7 mg6-OHDA into the right striatum and 7 mg 6-OHDA into the right substantianigra according to the atlas of known procedure.

Briefly, the animal is anesthetized with pentobarbital anesthesia(Sigma, 45 mg/kg, i.p.) and placed in stereotaxis instruments. Thesolution is prepared in a 0.2 mg/ml ascorbate saline and injected intoright striatum with a Hamilton syringe at a rate of 1 μl/min. Thestereotaxic co-ordinates are 1 mm anterior to bregma, 2 mm lateral frommidline, and 4.5 mm below the dura for striatum and 5.8 mm posterior tobregma, 1.6 mm lateral from midline, and 8 mm below the dura for SN partof brain with the incisor bar located 3.3 mm below the interaural lineon the non-dominant side.

Quantitation of Rotational Behavior:

On 14th day, after right striatum stereotaxic injection of 6-OHDA,animals are subjected to rotational behavior testing. Rats are injectedwith apomorphine hydrochloride subcutaneously (Sigma, 0.05 mg/kg) andafter 15 minutes from injection, contralateral turns are recorded for a30-minute period. In order to exclude the influence of apomorphine,rotational test is performed at last of the all other behavior test.

Stepping Test:

This test is used for measurement of akinesia. The rat is held with onehand by the experimenter fixing the hind limbs (slightly raising thetorso) and with the other hand fixing the forelimb that is not to bemonitored. In this way, the other forepaw has to bear the weight. Whenthe rats are moved with a speed of 90 cm per 5 s in forward and backwardalong the table, the free forelimb has to step with the movement of theexperimenter to keep balance. The steps taken to keep balance arerecorded as the adjusting steps. This is done for both the contralateraland ipsilateral forepaw and finally counted together. The numbers ofadjusting steps for both directions are counted.

Initiation Time:

The rats are pre-trained for two days to turn up a wooden ramp (1.1 m)into their home cage. During the test, the rat is held as per thestepping test. Time is measured until the rat initiated movement withthe forelimb is not fixed by the experimenter. This duration is definedas the initiation time and 180 s is used as the break-off point. Thetest is performed once a day for each forelimb on three consecutive daysand the mean of the three test sessions is calculated.

Postural Test:

The rat is held as described for the stepping test and then in a fastmovement tilted toward the side of the paw touching the table, whichcauses a loss of balance. The animal tries to regain balance with anadjusting step that is recorded by a scoring system ranging from 0 to 3:(0) no detectable muscle reaction, the rat falls onto the side; (1)clear forelimb reaction, but the rat cannot move limb under the bodytoward the center of gravity and thus still falls onto the side; (2)incomplete recovery of balance, i.e., the rat moves its limb under thebody but not yet fully into the center of gravity, and thus the forelimbis not aligned vertically to the body; further, the forepaw might not beplaced in a plain position on the table and digits might be crossed overeach other; (3) complete recovery of balance. The test is repeated sixtimes a day on both sides giving a maximum daily score of 18. Finalresults are expressed as average of the three-test day's score. Sixanimals repeat three times every day for three days, then meancalculates from 54 values for six rats in each group.

Disengage Time:

A blunt wooden probe touches the perioral region beneath the vibrissaeof the rat repeatedly at 1 s intervals when the rat is engaged in eatinga piece of milk chocolate. The latency of the orienting response, i.e.,turning of the head toward the stimulus, is recorded; an immediateresponse is scored as 1 s. Stimulation is discontinued if the animaldoes not respond within a period of 180 s. The test is performed once aday on each side over two days and the mean of the two subtests iscalculated.

All these tests (Rotational test, Stepping test, Initiation time,Postural test, Disengage time) are performed by a blind investigator,not aware of the treatment given to the animals.

Measurement of Dopamine and its Metabolites:

Animals are euthanized day after rotational test to exclude the effectof apomorphine. The striatal samples are separated and weighedimmediately after dissection and stored at −70° C. until assay. Thestriatal samples are sonicated in ice cold 0.2M perchloric acidcontaining 0.05% ethylenediamminetetraacetic acid (EDTA). Thehomogenates are immediately centrifuged at 10,000 rpm at 4° C. for 10minutes. The supernatants are filtered using 0.45-μm pore filters andare used for determination of dopamine (DA) and its metabolites3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) usinghigh-performance liquid chromatography with electrochemical detector.The exemplary mobile phase consists of a mixture of 0.1M citric acidmonohydrate, 0.1M sodium acetate, 7% methanol, 100 mM EDTA, and 0.01%sodium octane sulfonic acid. The flow rate of mobile phase is maintainedat 1 ml/min and the injection volume is 20 ml.

Statistical Analysis:

Data are expressed as mean±standard error of the mean (SEM). Multiplegroup comparisons are performed using one-way analysis of variance(ANOVA) follows by Dunnett's test. P<0.05 is considered statisticallysignificant.

Example 6: Randomized, Controlled Study Evaluating Compound 1 inSubjects with Mild to Moderate Alzheimer's Disease

Sixty patients with mild to moderate Alzheimer's disease willparticipate in this study. Half of the study subjects will take Compound1 orally, while the other half will take a placebo.

Study Design: Allocation: Randomized

Endpoint Classification: Safety/Efficacy Study

Intervention Model: Crossover Assignment

Masking: Double Blind (Subject, Caregiver, Investigator)

Primary Purpose Treatment

Primary Outcome Measures:

Alzheimer's Disease Assessment Scale-Cognitive Subscale 75 (ADAS-Cog 75)at Week 6 [Time Frame: Week 6]

12-item scale to assess severity of cognitive impairment in AD. Itemsinclude word recall, naming objects and fingers, following commands,constructional praxis, ideational praxis, orientation, word recognition,spoken language ability, comprehension of spoken language, word findingdifficulty in spontaneous speech, remembering test instructions, andconcentration/distractibility. Total score range from 0-75 with 75indicating worse cognition.

Secondary Outcome Measures:

Alzheimer's Disease Assessment Scale-Cognitive Subscale 75 (ADAS-Cog 75)at Week 3 [Time Frame: Week 3]

11-item scale designed to assess the severity of cognitive impairmentsin AD subjects. Items include word recall, naming objects and fingers,following commands, constructional praxis, ideational praxis,orientation, word recognition, spoken language ability, comprehension ofspoken language, word finding difficulty in spontaneous speech, andremembering test instructions. Total score range from 0-70 with 70indicating worse cognition.

Alzheimer's Disease Assessment Scale-Cognitive Subscale 70 (ADAS-Cog 70)at Week 6 [Time Frame: Week 6]

11-item scale designed to assess the severity of cognitive impairmentsin AD subjects. Items include word recall, naming objects and fingers,following commands, constructional praxis, ideational praxis,orientation, word recognition, spoken language ability, comprehension ofspoken language, word finding difficulty in spontaneous speech, andremembering test instructions. Total score range from 0-70 with 70indicating worse cognition.

Alzheimer's Disease Assessment Scale-Cognitive Subscale 70 (ADAS-Cog 70)at Week 6 [Time Frame: Week 6]

11-item scale designed to assess the severity of cognitive impairmentsin AD subjects. Items include word recall, naming objects and fingers,following commands, constructional praxis, ideational praxis,orientation, word recognition, spoken language ability, comprehension ofspoken language, word finding difficulty in spontaneous speech, andremembering test instructions. Total score range from 0-70 with 70indicating worse cognition.

Mean Clinical Global Impression—Improvement (CGI-I) Score at Week 6[Time Frame: Week 6]

CGI-I: 7-point clinician rated scale ranging from 1 (very much improved)to 7 (very much worse) Improvement is defined as a score of 1 (very muchimproved), 2 (much improved), or 3 (minimally improved) on the scale.Higher score=more affected.

Neuropsychiatric Inventory (NPI) Total Score at Week 3 [Time Frame: Week3]

Caregiver interview-based rating scale assessing 12 behavioraldisturbances occurring in dementia: delusions, hallucinations,agitation/aggression, depression/dysphoria, anxiety, elation/euphoria,apathy/indifference, disinhibition, irritability/lability, aberrantmotor behavior, appetite/eating, and sleep. Each symptom score derivedby frequency of symptoms*severity of symptoms (range 0-12). Totalscore=sum of symptom scores; range: 0-144 with higher score indicatinggreater behavioral disturbances.

Neuropsychiatric Inventory (NPI) Total Score at Week 6 [Time Frame: Week6]

Caregiver interview-based rating scale assessing 12 behavioraldisturbances occurring in dementia: delusions, hallucinations,agitation/aggression, depression/dysphoria, anxiety, elation/euphoria,apathy/indifference, disinhibition, irritability/lability, aberrantmotor behavior, appetite/eating, and sleep. Each symptom score derivedby frequency of symptoms*severity of symptoms (range 0-12). Totalscore=sum of symptom scores; range: 0-144 with higher score indicatinggreater behavioral disturbances.

Computerized Test Battery for Cognition (CogState) Tasks: Detection atWeek 3 [Time Frame: Week 3]

Assessment of the cognitive domain psychomotor function through yes orno responses within 5 minutes to 30 trials; score range: 0 to 3.69.Performance variable: speed of performance; average of the log 10 ttransformed reaction time for correct responses. Reaction times longerthan 5 seconds (log 10 [5000]) were excluded as reflecting responsesthat were abnormally slow.

Computerized Test Battery for Cognition (CogState) Tasks: Detection atWeek 6 [Time Frame: Week 6]

Assessment of the cognitive domain psychomotor function through yes orno responses within 5 minutes to 30 trials; score range: 0 to 3.69.Performance variable: speed of performance; average of the log 10 ttransformed reaction time for correct responses. Reaction times longerthan 5 seconds (log 10 [5000]) were excluded as reflecting responsesthat were abnormally slow.

In the case wherein the patient's status does improve, upon the doctor'sdiscretion the administration of the compounds may be given continuouslyor temporarily suspended for a certain length of time by the followingdesign.

Arms Assigned Interventions Experimental: Drug: Compound 1 Compound 1200 mg once daily for 1 week followed by 100 mg twice daily for 1 weekfollowed by 50 mg twice daily for 4 weeks Placebo Comparator: Drug:Placebo Placebo Placebo once daily for 1 week followed by placebo twicedaily for 5 weeks.Eligibility

Ages Eligible for Study: 55 Years to 85 Years

Genders Eligible for Study: Both

Criteria

Inclusion Criteria: Males or females, age 55-85

Diagnosis of probable Alzheimer's disease, consistent with criteria fromboth: 1) National Institute of Neurological and Communicable Disease andStroke and Alzheimer's Disease and Related Disorders Association(NINCDS-ADRDA) and 2) Diagnostic and Statistical Manual of MentalDisorders (DSM IV).

Mini-mental status exam score of 14-26 inclusive

Rosen-Modified Hachinski Ischemia Score of < or =4

Exclusion Criteria:

Diagnosis or history of other dementia or neurodegenerative disorders

Diagnosis or history of clinically significant cerebrovascular orcardiovascular disease

Subjects with pulmonary disease or evidence of clinically significantpulmonary symptoms

Example 7: Oral Formulation

To prepare a pharmaceutical composition for oral delivery, 100 mg of anexemplary Compound 1 was mixed with 100 mg of corn oil. The mixture wasincorporated into an oral dosage unit in a capsule, which is suitablefor oral administration.

In some instances, 100 mg of a compound described herein is mixed with750 mg of starch. The mixture is incorporated into an oral dosage unitfor, such as a hard gelatin capsule, which is suitable for oraladministration.

Example 8: Sublingual (Hard Lozenge) Formulation

To prepare a pharmaceutical composition for buccal delivery, such as ahard lozenge, mix 100 mg of a compound described herein, with 420 mg ofpowdered sugar mixed, with 1.6 mL of light corn syrup, 2.4 mL distilledwater, and 0.42 mL mint extract. The mixture is gently blended andpoured into a mold to form a lozenge suitable for buccal administration.

Example 9: Inhalation Composition

To prepare a pharmaceutical composition for inhalation delivery, 20 mgof a compound described herein is mixed with 50 mg of anhydrous citricacid and 100 mL of 0.9% sodium chloride solution. The mixture isincorporated into an inhalation delivery unit, such as a nebulizer,which is suitable for inhalation administration.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

What is claimed is:
 1. A method for alleviating, abating or amelioratingat least one symptom of, or reducing the risk of neurodegenerativedisease caused by neuronal damage comprising administering to a subjecta therapeutically effective amount of a composition consisting of acyclohexenone compound and one or more physiologically acceptablecarriers, and/or excipients, wherein the cyclohexenone compound is ofthe following structure:

wherein each of X and Y independently is oxygen, NR₅ or sulfur; R is ahydrogen or C(═O)C₁-C₈alkyl; each of R₁, R₂ and R₃ independently is ahydrogen, methyl or (CH₂)_(m)—CH₃; R₄ is 5 or 6-membered lactone,C₁-C₈alkyl, C₂-C₈alkenyl, C₂-C₈alkynyl, aryl, optionally substitutedwith one or more substituents selected from C₁-C₈ alkyl, C₂-C₈ alkenyl,C₂-C₈ alkynyl, C₃-C₈ cycloalkyl, and C₁-C₈ haloalkyl; m=1-12; andn=1-12; or a pharmaceutically acceptable salt, or solvate thereof,wherein said compound inhibits neuronal damage.
 2. The method accordingto claim 1, wherein said method inhibits β-Amyloid induced neuronaldamage in a subject.
 3. The method of claim 1, wherein saidneurodegenerative disease is selected from the group consisting ofAlzheimer's disease, Parkinson's disease, multiple sclerosis, strokesyndromes, and amyotrophic lateral sclerosis.
 4. The method of claim 3,wherein said neurodegenerative disease is Alzheimer's disease.
 5. Themethod according to claim 1, wherein said cyclohexenone compoundinhibits β-Amyloid induced neuronal damage in a subject.
 6. A method foralleviating, abating or ameliorating at least one symptom of a β-Amyloidinduced disease in a subject, comprising administering to the subject inneed a therapeutically effective amount of a composition consisting of acyclohexenone compound and one or more physiologically acceptablecarriers, and/or excipients, wherein the cyclohexenone compound is ofthe following structure:

wherein each of X and Y independently is oxygen, NR₅ or sulfur; R is ahydrogen or C(═O)C₁-C₈alkyl; each of R₁, R₂ and R₃ independently is ahydrogen, methyl or (CH₂)_(m)—CH₃; R₄ is 5 or 6-membered lactone,C₁-C₈alkyl, C₂-C₈alkenyl, C₂-C₈alkynyl, aryl, optionally substitutedwith one or more substituents selected from C₁-C₈ alkyl, C₂-C₈ alkenyl,C₂-C₈ alkynyl, C₃-C₈ cycloalkyl, and C₁-C₈ haloalkyl; m=1-12; andn=1-12; or a pharmaceutically acceptable salt, or solvate thereof,wherein said compound inhibits neuronal damage.
 7. The method accordingto claim 6, wherein the β-Amyloid induced disease is Alzheimer's diseaseor Down's syndrome.
 8. The method of claim 1, wherein said cyclohexenonecompound, or a pharmaceutically acceptable salt, or solvate thereof, isadministered orally, parenterally or intravenously.
 9. The method ofclaim 1, wherein said compound is isolated from Antrodia camphorata. 10.The method of claim 1, wherein R is a hydrogen, C(═O)C₃H₈, C(═O)C₂H₅, orC(═O)CH₃.
 11. The method of claim 1, wherein each of R₁, R₂ and R₃independently is a hydrogen, methyl, ethyl, propyl, butyl, pentyl,hexyl, heptyl, or octyl.
 12. The method of any one of claim 11, whereinR₁ is a hydrogen or methyl.
 13. The method of any one of claim 11,wherein R₂ is a hydrogen or methyl.
 14. The method of claim 1, whereinR₄ is C₁-C₈alkyl optionally substituted with one or more substituentsselected from C₁-C₈ alkyl, C₂-C₈ alkenyl, C₂-C₈ alkynyl, C₃-C₈cycloalkyl, and C₁-C₈ haloalkyl.
 15. The method of claim 14, wherein R₄is CH₂CH═C(CH₃)₂.
 16. The method of claim 15, wherein said compound is


17. The method of claim 1, wherein said subject is human.
 18. The methodof claim 6, wherein said cyclohexenone compound, or a pharmaceuticallyacceptable salt, or solvate thereof, is administered orally,parenterally or intravenously.